The long-term goal of this project is to understand the cellular and molecular mechanisms that lead to the structural changes associated with chronic bronchitis. This application continues our focus on oxidative stress-induced EGFR activation and mucus hypersecretion. During the current funding period we have confirmed the hypothesis of the original application, that reactive oxygen species (ROS) induce hyaluronan depolymerization releasing active bronchial TK that, in turn, results in pro-EGF processing, EGFR activation and downstream signaling that ultimately results in mucus cell proliferation and increased mucin secretion. However, in airway epithelial cells, EGFR is topologically separated from its ligands by apical junctional complexes (AJP): ligands are expressed in the apical membrane while receptors are predominantly localized to the basolateral membrane. The physical separation of growth factors from their receptors suggests that a regulated mechanism must exist that allows ligand-receptor interaction to occur only under certain conditions in which AJCs grant access to the basolateral side. We have found that oxidant-induced epithelial AJC junction disruption is mediated by RON, a tyrosine kinase receptor that is uniquely localized at the apical membrane of airway epithelia. The ligand for RON is the HGF-like macrophage stimulating protein (MSP), that is secreted as an inactive precursor that needs to be proteolytically cleaved to stimulate RON by a protease that, in the airways, is tissue kallikrein (TK). TK is present at the apical surface of airway epithelial cells, where it is bound and inactivated by hyaluronan (HA). ROS induced-HA depolymerization results in the release of active TK, a mechanism by which pro-MSP could be activated and thereby signal through its apical membrane receptor RON to disrupt AJC integrity allowing EGFR-ligand interaction and signaling. We will test the hypothesis that increased apical availability of active TK during oxidative stress is responsible for AJC disruption by activating pro-MSP and RON signaling, thereby allowing EGF receptor-ligand interaction, which ultimately results in airway mucous cell metaplasia and hypersecretion. This will be tested with the following aims: Aim 1 will test the hypothesis that TK, released from epithelial-bound HA by ROS, processes inactive pro-MSP to active MSP that binds and signals through RON to initiate epithelial AJC disruption (test of concept). Aim 2 will test the hypothesis that MSP-RON-dependent AJC disruption allows EGF-EGFR interaction that ultimately results in goblet cell proliferation and mucus hypersecretion (proof of concept). Aim 3 will test whether these mechanisms are operative in vivo in patients with chronic bronchitis (relevance of concept in airway epithelium). These studies will advance our knowledge on the mechanisms that lead to mucus hypersecretion and likely provide new tools for the prevention or/and treatment of chronic airway diseases, particularly cigarette smoke- induced chronic bronchitis. PUBLIC HEALTH RELEVANCE. Chronic bronchitis is an invalidating disease characterized chronic cough and frequent respiratory infections and results in frequent hospitalizations and poor quality of life. The most common cause of chronic bronchitis is exposure to cigarette smoke. We propose to use cells obtained from lung donors suffering from chronic bronchitis in combination with state of the art techniques to understand why mucous hypersecretion occurs and how can we intervene to prevent the disease or the progression of the disease.